Structure-function relationships in interphotoreceptor retinoid-binding protein (IRBP).
OBJECTIVE
Interphotoreceptor retinoid binding protein (IRBP) binds hydrophobic ligands in the retina. The polypeptide consists of 1230 amino acids in four 300 amino acid long repeats. We asked whether each of the four repeats can bind one retinoid or fatty acid analog. Our rationale was to make protein variants from the human cDNA bearing one or more of the repeats and examine binding capacities and dissociation constants.


METHODS
Proteins were characterized by SDS-PAGE, western blotting, N-terminal sequencing, and CD spectroscopy. Binding properties with all-trans-retinol and 16-anthryloxy-palmitic acid (16-AP) were characterized by ligand fluorescence enhancement and curve fitting.


RESULTS
Binding capacities varied according to the length of each protein. Each repeat possesses the capability of binding retinol and 16-AP.


CONCLUSIONS
The data contrast with the idea that two or more repeats are needed to bind one molecule of ligand. Each repeat binds a retinoid and fatty acid analog, suggesting that each has multiple ligand binding sites or one binding site with affinity for different ligands. Last, these data fit well with the current model of multiple binding sites in IRBP derived from quadruplication of an ancestral monomeric binding protein.
Lin
Z Y
ZY
Department of Ophthalmology, Emory University, Atlanta, GA 30322, USA.
Li
G R
GR
Takizawa
N
N
Si
J S
JS
Gross
E A
EA
Richardson
K
K
Nickerson
J M
JM
D000818:Animals; D002478:Cells, Cultured; D002942:Circular Dichroism; D005136:Eye Proteins; D006801:Humans; D007313:Insecta; D016296:Mutagenesis; D010169:Palmitic Acids / Q000378:metabolism*; D017354:Point Mutation; D012177:Retinol-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014801:Vitamin A / Q000378:metabolism*; D000099214:Retinol-Binding Proteins, Interstitial
D005136:Eye Proteins; D010169:Palmitic Acids; D012177:Retinol-Binding Proteins; D014801:Vitamin A; D000099214:Retinol-Binding Proteins, Interstitial; C047265:16-(9-anthroyloxy)palmitic acid